Our overall goal of these studies is to elucidate the mechanism(s) underlying Toll-like receptor (TLR) mediated immune suppression during stress. Physical and psychological stress can alter the immune system in both humans and animals. Stress is also a known risk factor for numerous human diseases. TLR mediated signaling pathways play a critical role in the induction of immune response. TLRs mediate the immune response to stress;however, the mechanisms remain to be elucidated. In order to study the mechanisms of stress-induced immune responses and to design strategies for therapeutics, we established an animal model for restraint stress to study immune suppression. Using this model we have revealed that restraint stress of mice modulates the immune system through a cell apoptotic mechanism. We found that TLR4 deficient mice are resistant to stress-induced splenocyte reduction. TLR4 typically interacts with high mobility group box-1 protein (HMGB1). Our preliminary data indicate that stress enhances plasma HMGB1 levels through TLR4. The phosphoinositide 3-kinases (PI3K) and their downstream substrate Akt can modulate TLR-mediated responses by decreasing inflammation and inhibiting apoptosis. We have shown that TLR4 deficiency in mice induces protection in splenocyte numbers which is mediated through PI3K. Collectively, our data suggest that TLR4 and PI3K/Akt signaling play a role in immune responses during stress. At present, we do not understand the precise role of TLR4-mediated signaling in stress-induced lymphocyte apoptosis, nor do we understand the precise role of TLR4 in negative regulation of the PI3K/Akt signaling in immune suppression during stress. Our hypothesis is that modulation of TLR4 and the PI3K/Akt signaling pathways determines the fate of lymphocytes in response to stress. We propose the following aims to test this hypothesis. Aim 1 will define the role of TLR4-mediated apoptotic signaling pathways following stress. To elucidate whether TLR4 activates apoptotic signaling pathways during stress, we will first investigate the effects of TLR4-mediated signaling on lymphocyte apoptosis, including CD4+ and CD8+ T cell apoptosis. We will then examine whether TLR4 deficiency activates PI3K/Akt signaling in stress-induced lymphocyte apoptosis. Aim 2 will define the role TLR4 negative regulation of PI3K/Akt signaling in stress-induced immune suppression. To examine whether TLR4 deficiency activates PI3K/Akt signaling, resulting in immune protection, TLR4 knockout mice and wild type mice will be administered PI3K inhibitors, and then subjected to stress for different time periods. We will define the role of TLR4 mediated signaling in stress-induced immune suppression, and then determine whether TLR4 deficiency results in activation of PI3K/Akt signaling. In addition, we will determine the role of HMGB1 and a multifunctional adaptor 2-arrestin 2 in TLR4-mediated PI3K/Akt signaling following stress. Our studies should lead to our understanding of the effects of TLR4-mediated signaling pathways on the immune system, providing the opportunity to develop novel strategies and therapeutics for immune suppression. PUBLIC HEALTH RELEVANCE: Psychological and physical stress can have pronounced effects on the immune system. Historically this affect was attributed to the release of stress hormones although other factors appear to play an important role. Using a mouse model of physical restraint stress we have investigated some of the molecular mediators which may account for the connection between stress and the immune system. This research will examine the mechanisms whereby the immune system is influenced by stress with the hope of leading to the development of specific treatments or drugs which in turn may ameliorate this sometimes deleterious interaction.